Morphogenetic study during cell division in Euplotidium itoi (ciliata, hypotrichida)

Europ. J. Protisto!' 31,286-291 (1995) August 25, 1995

European Journal of

PROTISTOLOGY

Morphogenetic Study During Cell Division Euplotidium itoi (Ciliata, Hypotrichida)

.

In

M. Anita Giambelluca, Simone Gabrielli, Fabrizio Erra, and Giovanna Rosati Dipartimento di Scienze dell' Ambiente e del Territorio, Universita di Pisa, Pisa, Italy

SUMMARY The present study, performed by means of light and scanning electron microscopy, is the first report in which morphogenetic processes are analyzed in detail in a member of the Euplotidium genus. From our observation it arises that E. itoi shares many morphogenetical features with members of the Euplotidae family, particularly with Euplotes. The replication band forms at the distal end of each macronuclear piece, proceeds proximally and disappears as the fusion of the pieces occurs. The parental adoral zone of membranelles (AZM) and paroral membrane (PM) are inherited by the proter; the corresponding structures form de novo, probably from separated anlagen, in the opisthe. New sets of cirri are formed for both products of division. Only as far as the proliferation of dorsal kineties is concerned does E. itoi resemble the genus Diophrys rather than Euplotes.

Introduction The genus Euplotidium, established by Noland [9] in 1937, is at present comprised of six species [1]. A morphological analysis at the ultrastructural level has been performed on Euplotidium itoi [7], while information about the other species is based only upon light microscope studies [3, 6, 8,9, 12, 13]. As for divisional morphogenesis, there exists, as far as we know, very little data on nuclear [8] and cortical [4] events in E. arenarium. The scarceness of studies concerning Euplotidium, in comparison with other common ciliates, is probably due to the poor adaptability of the members of this genus to growth in laboratory cultures. Moreover, judging from our experience and from what can be detected by the available literature [12] all the species described have a restricted well-characterized ecological niche in which, only in rare cases, they can be found in abundance. We were successful in growing E. itoi in our lab under well controlled, although certainly not optimal conditions (the cell cycle for example is unusually long). In any case, as a contribution, the morphogenesis during binary fission of E. itoi has been analyzed by means of light and scanning electron microscopes. The results obtained are reported in this paper. 0932-4739-95-0031-0286$3.50-0

Material and Methods Euplotidium itoi was collected from sea water along the shore located south of Leghorn (Italy) in an area characterized by a rocky platform in which there are interconnected tidal pools linked to the open sea by channels. The specimens were cultured in the laboratory at 20-24 °C in pasteurized original sea water containing a small amount of sand grains, periodically enriched with the green flagellate Dunaliella salina and the diatom Pheodactilum tricornutum. In these conditions (the most productive among those tested) the cell cycle lasts about 48 h. This unusual length causes a high asynchrony among the cells of the same culture and makes it very difficult to find all morphogenetic stages among silver stained cells. For this reason most data have been obtained by means of scanning electron microscopical observations on synchronized cells. To obtain specimens which were as synchronous as possible groups of 15 - 20 dividers were isolated and fixed separately at different times. The first group was fixed at the time (determined by the Feulgen procedure) that the reorganization bands would approximately appear in the macronuclear pieces (see results). The other groups were subsequently fixed at different intervals which, as the end of the process approached, became shorter. The last group was fixed soon after the accomplishment of the new binary fission. For scanning electron microscopy (SEM) specimens were processed as described elsewhere [14]. © 1995 by Gustav Fischer Verlag, Stuttgart

Morphogenesis in E. itoi . 287 In the drawings, mainly based on SEM observations, parental cirri are shown by their contour while the new cirri are filled in.

Results 1. Non-dividing Stage (Fig. La, b)

The morphology of E. itoi in the non-dividing stage has previously been described in detail [7, 14] so it is briefly referred to here. On the dorsal surface, there are 5 kineties, while on the ventral surface, besides the huge oral cavity with the adoral zone of membranelles (AZM) and the paroral membrane (PM), there are 12 frontoventral cirri (FVC), 6 transversal cirri (TV) and 1 left marginal cirrus (MC). The cirral pattern, as also reported for other members of the Euplotidae family [1], is sometimes varied: some specimens have 13 VFC and 7 TC, while others have 2 MC arising very close to each other. The nuclear apparatus consists of two elongated macronuclear pieces and four micronuclei. 2. Morphogenesis (Figs. 2-8, 9-14)

The whole process can be outlined as follows: A. (Figs. 2, 9). The first detectable morphological events are at the macronuclear level: the two macronuclear pieces move closer to each other and a replication band appears at the terminal end of each one. The replication bands move toward the upper ends of the macronuclear pieces which now tend to come into contact (Fig. 2b). Meanwhile, the oral primordium (OP) becomes visible on the ventral surface to the left of and posterior to the parental oral apparatus (POA) and anterior to the MC (Figs. 2a, 9). At the same time new sets of cirri develop along 6 streaks to the right of the POA and anterior to the parental TC (Figs. 2a, 9). Unlike the cirral number, the number of cirral streaks is the same in all the specimens examined. B. (Figs. 3, 10). The two macronuclear pieces, still not completely fused, are now condensed and shortened while the replication bands approach their term-

inal ends (Fig. 3b). The OP and the cirral anlagen fields grow up maintaining the same positions; two distinct groups of cilia arise far from the OP, externally to the right margin of the POA. These probably represent the opisthe's primordia of the paroral cirrus (PC) and the PM (Figs. 3a, 10). In the same picture another ciliary primordium can be seen (although faintly) anterior to the two mentioned above and internal to the margin of the buccal cavity. As the parental PM is still well formed beneath the thin lamina that typically covers it in the non dividing stage [7], it probably represents the proter's PC primordium. C. (Figs. 4, 11) The two macronuclear pieces have completely fused at their anterior ends and the replication bands are no longer recognizable (Fig. 4b). The OP sinks into a pouch that forms on the ventral surface, posterior to the cavity of the POA. The cilia begin to align into membranelles (Figs. 4a, 11). D. (Figs. 5, 12-14). The macronucleus begins to divide in two (Fig. 5b). At the same time the new oral cavity becomes increasingly wider and deeper; the primordia of the proter's and opisthe's MC can be seen arising at its left margin. By this time the opisthe's PM has already reached its final position (Figs. Sa, 12, 13). On the dorsal surface the kineties are separated into two distinct sets, both with more numerous cilia than the kineties in the non-dividing stage (Figs. 5b, 14). E. (Figs. 6, 15-17). The macronucleus consists of two condensed pieces (Figs. 6b, 17). During this stage the OP gradually assumes its final shape and organization. It increases in length and migrates forward along the left side of the organism. The majority of parental cirri have not yet been reabsorbed and the new ones are still crowded along the ventral surface (Figs. 6a, 15). A lateral shifting of the opisthe with respect to the proter is also evident on the dorsal side (Figs. 6b, 16) where an initial divisional furrow is visible on the left. E (Figs. 7, 18, 19). The macronuclear pieces migrate in opposite directions (Fig.7b). The furrow is now also evident on the right side of the ciliate; the reabsorption of old cirri and the positioning of the new sets has not yet been accomplished. The opisthe's oral apparatus is fully formed and well positioned

Fig. la, b. Euplotidium itoi in non-dividing stage. - Fig. 1a. Ventral view. - Fig. lb. Dorsal view. AZM =adoral zone of membranelles; EB =epixenosomal band; DK =dorsal kineties; FVC =frontoventral cirri; Ma =macronuclear pieces; MC =marginal cirrus; Mi = micronuclei; PM = paroral membrane; TC = transverse cirri. Bar = 30 11m. - Figs. 2-7. Morphogenesis in E. itoi: stages A-F. - Fig. 2a, b Ventral and dorsal view during A stage. Formation of oral primordium (OP) and anlagen of frontoventral and transverse cirri (CA), appearance of replication bands of macronuclei (RB). - Fig. 3a, b. Ventral and dorsal view of stage B. Thin arrow indicates the primordia of the opisthe's paroral cirrus and paroral membrane. Large arrow indicates the primordium of the proter's paroral cirrus. Macronuclear pieces are closer to each other and the replication bands proceed towards their terminal ends. - Fig. 4a, b. Ventral and dorsal view during stage C. Alignment of membranelles in OP begins. The fusion of macronuclear pieces is finished.- Fig. Sa, b. Ventral and dorsal view during stage D. The differentiation of cirri and AZM is in progress. Formation of marginal cirri anlagen (MCA). The dorsal kineties are separated in two thickened sets. The macronucleus begins to divide. - Fig. 6a, b. Ventral and dorsal view during stage E. The OP assumes its final organization. Parental cirri begin to be reabsorbed. The macronucleus consists of two condensed pieces. - Fig. 7a, b. Ventral and dorsal view during stage F,that is just before the daughter cells' separation. The reabsorption of parental cirri and the positioning of the new ones is not jet accomplished. Bar = 30 11m. - Fig. 8a, b. Stage G. - Fig. 8a. Ventral view of a just separated opisthe. Some transversal cirri (TC) are still unreabsorbed. - Fig. 8b. Proter, dorsal view. Only one macronuclear piece is present. Bar =30 11m.

~

288 . M. A. Giambelluca, S. Gabrielli, F. Erra, and G. Rosati

Mi

Ma

Te

1b

1a

Sa

5b

RB

2a

3a

2b

6a

6b

3b

7a

4a

4b

8a

7b

8b

Figs. 9-14. SEM pictures of E. itoi during morphogenetical stages A-D. - Fig. 9. Stage A. The or is evident on the ventral surface. The cirral anlagen arise along six streaks. The white arrow indicates the two last, less developed, x 1100. - Fig. 10. Stage B. The black arrows point to the opisthe's primordia of the paroral cirrus and paroral membrane. The white arrow indicates the probable prater's paroral cirrus, x 1600. - Fig. 11. Stage C. The oral primordium sinks into a pouch of the ventral surface, x 2300. -Fig. 12. Advanced stage C. The oral primordium lies deep in the ventral pouch, x 1100. - Figs. 13-14. Stage D. - Fig. 13. Close-up of the oral primordium. The primordia of marginal cirri of both proter and opisthe are evident at its left margin, x 3000. - Fig. 14. Dorsal view showing the kineties separated in two distinct sets, x 700. MCr = marginal cirri primordia; OP = oral primordium; OPM = opisthe paroral membrane; PPM = prater paroral membrane.

290 . M. A. Giambelluca, S. Gabrielli, F. Erra, and G. Rosati

Figs. 15-19. SEM pictures of morphogenetical stages E-G. - Figs. 15-16. Ventral and dorsal view during stage E: note the lateral shifting of the opisthe with respect to the proter. Fig. 15, x 1000, Fig. 16, x 700. - Fig. 17. During stage E the macronucleus consists of two condensed pieces, x 340. - Figs. 18-19. Ventral and dorsal view during stage F. The divisional furrow is evident on both sides of the ciliate. Fig. 18, x 600, Fig. 19, x 700. - Figs. 20-21. Just separated daughter cells (stage G). Fig. 20. An opisthe from the ventral side: parental transversal cirri (arrow) are not completely reabsorbed, x 900. - Fig. 21. Dorsal view of a proter, the dorsal kineties are not delimited by the ribs typical of non-dividing stage, x 900.

Morphogenesis in E. itoi . 291

(Figs. 7a, 18). Figs. 7b and 19 show the last step of cell division in dorsal view. G. (Figs. 8,20,21). The recently divided E. itoi have only one macronuclear piece (Fig. 8b). Fig. 20 shows an opisthe from its ventral side: some TC are still not reabsorbed. In Fig. 21 the proter's dorsal side can be seen soon after division: the kineties are not bordered by the ribs typically present during the non-dividing stage.

Discussion E. itoi shares many morphogenetic characteristics with members of the Euplotidae family and appears more closely related to members of the genus Euplotes than other euplotid-like genera. As in Euplotes [2, 10, 15] the parental AZM and PM are inherited apparently unchanged by the proter, while the opisthe AZM anlage develops to the left and posterior to the parental cavity, independent from parental AZM. New sets of cirri are formed for both products of division. Frontoventral and transverse cirri develop from six streaks that have respectively 3,3,3,3,2,3 cirri, differently from E. arenarium in which the streaks are five [4]. The proter's PC arises very close to parental PM, so it is reasonable to suggest that it derives from this pre-existing ciliary structure. On the other hand the primordia of the opisthe's PC and PM appear situated rather far from developing OP. So on the basis of our observations, their direct connection with OP cannot be affirmed. The left marginal cirrus of both daughter organisms develops from anlagen which later form along the left edge of the body with respect to the other cirral primordia. Dorsal anlagen develop within each of the existing dorsal kineties as two separate sets: for this trait E. itoi resembles members of the genus Diophrys [5, 11] rather than of the genus Euplotes. The morphogenetic process is fully accomplished after the separation of proter and opisthe: recently divided Euplotidium have only one macronuclear piece, old cirri are still unreabsorbed and the kineties are not bordered by the typical cortical ribs present in the non dividing stage.

References 1 Curds C. R. and Wu I. C. H. (1983): A review of Euplotidae (Hypotrichida, Ciliophora). Bul!. Br.Mus. nat. Hist. (Zoo!.), 44, 191-247. 2 Fleury A. (1991): Dynamics of the cytoskeleton during morphogenesis in the ciliate Euplotes. I. Basal bodies related microtubular system. Europ. J. Protisto!., 27, 99114. 3 Hartwig E. (1980): The interstitial ciliates of Bermuda with notes on their geographical distribution and habitat. Cah. Bio!. Mar., 21, 409-441. 4 Hill B. F. (1980): Euplotidium arenarium Magagnini & Nobili, 1964: cortical morphogenesis associated with cell division (Ciliophora, Hypotrichida). J. Protozoo!., 27, 12A. 5 Hill B. F. (1981): The cortical morphogenetic cycle associated with cell division in Diophrys Dujardin, 1841 (Ciliophora Hypotrichida). J. Protozoo!., 28, 215-221. 6 Ito S. (1958): Two new species of marine ciliate. Euplotidium itoi sp. nov. and Gastrocirrhus trichocystus sp. nov. Zoo!' Mag. Tokyo, 67, 184-187. 7 Lenzi P.and Rosati G. (1993): Ultrastructural study of Euplotidium itoi (Ciliata, Hypotrichida). Europ.J. Protisto!., 29, 453-461. 8 Magagnini G. e Nobili R. (1964): Su Euplotes woodruffi Gawe su Euplotidium arenarium n. sp. (Ciliatea, Hypotrichida). Mon. Zoo!' Ita!., 72, 178-202. 9 Noland L. E. (1937): Observation on marine ciliates of the gulf coast of Florida. Trans. Am. Microsc. Soc., 56, 160171. 10 Ruffolo J.J. jr. (1976): Cortical morphogenesis during the cell division cycle in Euplotes: an integrated study using light optical, scanning electron and transmission electron microscopy. J. Morpho!., 14, 489-528. 11 Song W. and Wilbert N. (1994): Morphogenesis of the marine ciliate Diophrys oligotrix Borror 1965, during the cell division (Protozoa, Ciliophora, Hypotricha). Europ. J. Protisto!., 30, 38-44. 12 Tuffrau M. (1985): Une nouvelle espece du genre Euplotidium Noland 1937: Euplotidium prosaltans n. sp. (Cilie hypotriche). Cah. Bio!. Mar., 26, 53-62. 13 Vacelet E. (1961): La faune infusorienne des "Sables aamphioxus" des environs de Marseille. Bul!. Inst. Ocean Monaco, 3, 1-12. 14 Verni F. and Rosati G. (1990): Peculiar epibionts in Euplotidium itoi (Ciliata, Hypotrichida). J. Protozoo!., 37, 337-343. 15 Wise B. N. (1965): The morphogenetic cycle in Euplotes eurystomus and its bearing on problems of ciliate morphogenesis. J. Protozoo!., 12, 626 -648.

Key words: Divisional morphogenesis - Hypotrich ciliate - Euplotidium Giovanna Rosati, Dipartimento di Scienze dell' Ambiente e Territorio, Universita di Pisa, via A. Volta n. 4, 56126 Pisa, Italy